Symposium

Symposium 37 Toward the understanding and control of neural functions via transcriptional regulation シンポジウム37 転写制御を介した神経機能調節研究の新潮流

SY37-1 The Trisynaptic Disinhibitory Network linking Mushroom Body Parallel Circuits Induces Memory Consolidation in Drosophila ショウジョウバエ記憶中枢並列回路をつなぐ脱抑制回路が、遺伝子発現誘導を介した記憶固定化を誘導する Hirano Yukinori（平野恭敬） Dept. of Phama. Med., Univ. of Kyoto Memory consolidation is augmented by repeated learning following rest intervals, which is known as the spacing effect. Although the spacing effect has been associated with cumulative cellular responses in the neurons engaged in memory, here, we report the neural circuit-based mechanism for generating the spacing effect in the memory-related mushroom body (MB) parallel circuits in Drosophila. In an olfactory spaced training paradigm, one of the parallel circuits, consisting of　γmain neurons, was progressively inhibited via dopamine. This Inhibition resulted in reduced activity of a postsynaptic GABAergic neuron that, in turn, disinhibited a neuron triggering gene expression in the different MB parallel circuit consisting of αβs neurons for memory consolidation. Thus, the spacing effect-generating neurons and the neurons engaged in memory reside in the distinct MB parallel circuits, which are linked by the disinhibitory network, suggesting that the spacing effect can be a consequence of evolved neural circuit architecture.		SY37-2 Regulation of SRF transcriptional cofactor MKL/MRTF by neuronal activity and its function in neurons 神経活動によるSRF転写コファクターMKL/MRTFの制御とその役割 Tabuchi Akiko（田渕明子）¹，伊原大輔¹，福地守^1,2，田邉広樹¹，阪上洋行³，大塚稔久⁴，津田正明¹ ¹L ab. of Mol. Neurobiol., Grad. Sch of Med. and Pharm. Sci., Univ. of Toyama, Toyama, Japan ²Lab. of Mol. Neurosci., Fac of Pharm., Takasaki Univ. of Health and Welfare, Gunma, Japan ³Dept. of Anat., Kitasato Univ. Sch. of Med., Kanagawa, Japan ⁴Dept. Biochem., Fac. of Med/Grad. Sch. of Med., Univ. of Yamanashi, Yamanashi, Japan Megakaryoblastic leukemia/myocardin-related transcription factor (MKL/MRTF) family members are serum response factor (SRF) coactivators. We have found that MKL/MRTF is highly expressed in the brain and regulates dendritic morphology. However, the precise localization and synaptic function of MKL/MRTF in neurons remained unresolved. Thus, we generated and tested new antibodies that distinctly and specifically recognize endogenously expressed MKL1/MRTFA and MKL2/MRTFB proteins in neurons. Using these antibodies, we demonstrate that MKL1/MRTFA and MKL2/MRTFB are localized at synapses. RNAi experiments revealed that knock-down of MKL reduced the percentage of mushroom- or stubby-type spines in cultured cortical neurons. Therefore, MKL are involved in dendritic spine maturation. Next, we tested whether MKL1/MRTFA and MKL2/MRTFB translocate from synapses to nucleus in cortical neurons. We found that membrane depolarization transiently induced nuclear translocation of MKL2/MRTFB. The nuclear translocation was also observed by stimulation with bicuculline/4AP, suggesting that synaptic activity mediates this process. Inhibitors for NMDA receptors and L-voltage dependent Ca²⁺ channels completely blocked the nuclear translocation. Taken together, these findings suggest that, like CREB-regulated transcriptional coactivator 1 (CRTC1), MKL2/MRTFB is transported from synapses to nucleus in a neuronal activity-Ca²⁺ signaling-dependent manner and thereby regulates gene expression. Since nucleotide substitutions in the MKL1/MRTFA and MKL2/MRTFB genes are found in patients with neurological disorders, the studies on MKL/MRTF may, at least in part, contribute to better understanding of not only health but diseases in the brain.

SY37-3 Transcription factor Npas4 regulates neural function in the normal and disease brain 健常脳および病態脳における転写因子Npas4による神経機能の制御機構 Takahashi Hiroo（高橋弘雄）¹，朝比奈諒¹，藤岡正行¹，坪井昭夫^1,2 ¹Lab for Mol Biol of Neural System, Center of Front Medicine, Nara Med Univ, Kashihara, Japan ²Grad Sch of Front Biosciences, Osaka Univ, Suita, Japan After ischemic stroke, most of neurons die in the infarct core region, leading to induction of the severe brain dysfunction. However, a mechanism that regulates neuronal death or survival around the infarct area after stroke remains unknown. To investigate the profile of gene expression, induced immediately after middle cerebral artery occlusion (MCAO) in mice, RNA samples were prepared from the control and ischemic sides of neocortex at 2 h after stroke, and subjected to RNA-Seq analysis. We found by in situ hybridization that 25 genes were expressed more abundantly in the ischemic side than the control side of cortex. Notably, the activity-dependent transcription factor gene, Npas4, was expressed immediately in both excitatory and inhibitory neurons around the infarct cortex. In the normal brain, we previously reported that Npas4 is expressed in a subset of olfactory bulb interneurons following sensory experience and regulates the dendritic spine formation (Cell Rep 8, 843, 2014). Interestingly, in Npas4 knockout mice, primary-cultured neurons under oxygen-glucose deprivation (OGD) showed an increase of cell death, while its overexpression in the cultured neurons reduced the cell death, compared with the control. Npas4 knockout mice following MCAO showed the increase of the infract size and severe behavioral dysfunction, whereas its overexpression in the wild-type mice before MCAO reduced the infract size, compared with the control. Our findings suggest that Npas4 expression, induced immediately after stroke, plays a crucial role in neuronal survival in the disease brain.		SY37-4 Memory-related activity-regulated transcripts revealed by single cell RNA-Seq analysis 認知活動により発現誘導される遺伝子の単一細胞レベルでの網羅的解析 Okuno Hiroyuki（奥野浩行） B iochem. & Mol. Biol., Kagoshima Univ. Grad. School of Medical and Dental Sciences Immediate early genes (IEGs), such as c-fos and egr-1, are widely used as the reliable molecular markers for activated neurons in the brain. Recent studies using opto/chemogenetics have suggested that IEG-positive cell ensembles possibly function as memory traces in the brain. However, it still remains unknown how IEGs regulate synaptic plasticity and circuit reorganization that underlie various cognitive functions including learning and memory. We have been focusing on a particular IEG named Arc because Arc plays critical roles in synaptic plasticity and homeostatic scaling by regulating AMPA receptor (AMPA-R) trafficking. We previously investigated molecular mechanisms of rapid synaptic activity-dependent expression of Arc and generated various activity-dependent gene expression reporter systems based on the Arc’s activity-dependent enhancer SARE (Kawashima et al., PNAS 2009; Nat Methods 2013, Okuno et al., Cell, 2012). Using these tools, we here report that a novel single-cell RNA-Seq analysis to identify activity-regulated transcripts in hippocampal neurons activated during performing memory tasks. This novel approach allows to directly compare activated and non-activated neurons with information of cell morphology and cell location, in the same animals. Using this method, we have identified many novel activity-regulated genes. Characterization and further analyses for these newly identified genes are ongoing.

SY37-5 Regulatory Mechanism of Neural Stem Cells Revealed by Optical Manipulation of Gene Expressions 遺伝子発現の光操作技術と幹細胞研究への応用 Imayoshi Itaru（今吉格） Graduate School of Biostudies, Kyoto Univ. The mammalian brain consists of a complex ensemble of neurons and glial cell s. Their production during development and remodeling is tightly controlled by various regulatory mechanisms in neural stem cells. Among such regulations, basic helix-loop-helix (bHLH) factors have key functions in the self-renewal, multipotency, and fate determination of neural stem cells. Here, we highlight the importance of the expression dynamics of bHLH factors in these processes. We propose the multipotent state correlates with oscillatory expression of several bHLH factors, whereas the differentiated state correlates with sustained expression of a single bHLH factor. We also developed a new optogenetic method that can manipulate gene expressions in neural stem cells by light. We used this technology to manipulate the growth and fate-determination of neural stem cells. I also introduce various applications of light-induced control of gene expressions in broad fields of biology.